This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; methods of making the polynucleotides and polypeptides, and their variants and derivatives; antibodies directed toward the polypeptides, their variants and derivatives; and uses of the polynucleotides, polypeptides, variants, derivatives and antibodies. In particular, in these and in other regards, the invention relates to novel human extracellular matrix polypeptides (designated RG1), polynucleotides which encode these polypeptides, antibodies directed toward these polypeptides, and antisense polynucleotides that block RG1 expression.
Prostate cancer is a frequently occurring disease in man, in that it is found in about one third of men over the age of 45. There is evidence for both genetic and environmental causes, with the majority of cases probably being the result of a combination of both factors. Studies of familial cancer have suggested that genetic predisposition plays a role in about 5-10% of all prostate cancers, and in about 45% of cases in men younger than 55.
There is evidence that prostate cancer develops as a multi-step disease, with one of the precursor lesions being prostatic intraepithelial neoplasia (PIN). Early stages of the disease are androgen dependent, while later stages are hormone independent. A proliferative disorder of the prostate known as benign prostatic hyperplasia is often detected clinically but is probably not a stage in the development of cancer. It is, however, frequently associated with prostate cancer. Cancers in the prostate are often multifocal, generally slow growing, and heterogeneous. Late stage cancers frequently metastasize to the lymph nodes and to the bone.
Prostate cancer is usually diagnosed by physical examination and by serum levels of prostate specific antigen (PSA). Radical prostatectomy is the treatment of choice for localized disease. Advanced metastatic disease is treated currently by androgen ablation induced by orchiectomy or treatment with GnRH (gonadotrophin releasing hormone), and by anti-androgen therapy. However, advanced disease almost invariably becomes hormone resistant and there is no cure for progressive disease. Moreover, there are serious side effects associated with both radical prostatectomy and androgen ablation therapy. These include a high risk of incontinence and impotence associated with radical prostatectomy and bone fractures and osteoporosis associated with androgen ablation therapy.
There is, therefore, a considerable need for new therapeutic approaches for both early and late stage prostate cancer. There is also a significant need for new diagnostic agents, in particular agents that can discriminate stages of the disease, as this significantly influences the treatment options. For example, if disease has progressed beyond the prostate and has metastasized to the lymph nodes, radical prostatectomy is not undertaken as it has no effect on progression, but may have significant unwanted side effects. An agent that could detect metastasis, in vivo, would have considerable value.
Changes in the expression of specific proteins have been demonstrated in prostate cancer including abnormal p53 expression in late stage prostate cancer, reduced levels of TGF-xcex2 receptors, reduced levels of E-cadherin, C-Cam (a cell adhesion molecule), and several integrins. The expression of the oncogene bcl-2 is strikingly elevated in late stage androgen independent tumors, and prognosis for patients expression bcl-2 at elevated levels is relatively poor. While the previously mentioned changes in gene expression are well documented, no changes in expression have been identified that have been demonstrated to be causative for the disease. It would, therefore, be useful to identify new proteins whose expression is linked to the presence or development of prostate tumors which could serve as molecular targets for prostate cancer diagnosis and therapy.
This invention discloses a new homologue to a superfamily of extracellular matrix proteins. This homologue, named RG1 is expressed in prostate tissue and may be over-expressed in prostate tumors.
The extracellular matrix is a complex meshwork of collagen and elastin, embedded in a viscoelastic ground substance composed of proteoglycans and glycoproteins. The matrix exists as a three dimensional supporting scaffold that isolates tissue compartments, mediates cell attachment and determines tissue architecture (Bissel et al., J. Theor. Biol. 99:31-68, 1982; Carlson et al., Proc. Natl. Acad. Sci. USA 78:2403-2406, 1981). The matrix acts as a macromolecular filter (Hay, E. D., Cell Biology of Extracellular Matrix, New York, Plenum Press, 1982) and also influences cytodifferentiation, mitogenesis, and morphogenesis (Gospodarowiczs, D., Cancer Res. 38:4155-171, 1978). The biochemical interactions between normal cells and the matrix may be altered in neoplasia, and this may influence tumor proliferation. Tumor cells can interact with the matrix in different ways. First, tumor cells can attach to the matrix via specific plasma membrane receptors (Terranova et al., Cancer Res. 42:2265-2269, 1982). Second, degradation of the matrix is mediated by a cascade of enzymes that are contributed by the tumor cell and the host (Eisen et al., Bioch. Biophys. Acta 151:637-645, 1968). Third, in differentiated areas of the tumor, tumor cells may synthesize and accumulate matrix or induce the host cell to accumulate excessive matrix (Brownstein et al., Cancer 40: 2979-2986, 1977).
RG1 shows homology to a superfamily of extracellular matrix proteins, encoded by the Mindin/F-spondin genes. The gene family is united by two conserved spondin domains, FS1 and FS2, near the amino terminus and at least one thrombospondin type 1 repeat (TSR1) at the carboxy terminus (Shimeld, S. M., Mol. Biol. Evol. 15(9): 1218-1223, 1998). The TSR motif was originally found in the vertebrate extracellular matrix proteins (Bornstein, P., J. Cell Biol. 130:503-506, 1995) and has subsequently been found in several other extracellular matrix proteins. There are several lines of evidence that TSR""s mediate cell adhesion and play a key role in tumorigenesis. For example, it has been demonstrated that proteolytic fragments of thrombospondin that contain the TSR""s, and synthetic peptides having sequences corresponding to the TSR region of thrombospondin, promote tumor cell adhesion and metastasis (Prater et al., J. Cell Biol. 112:1031-1040, 1991; Tuszynski and Nicosia, BioEssays 18:71-76, 1996), have anti-angiogenic activity (Tolsma et al., J. Cell Biol. 122:497-511, 1993) and inhibit platelet aggregation and melanoma metastasis (Tuszynski et al., J. Cell Biol. 116:209-217, 1992).
Currently, the members of this superfamily include a gene in Caenorhabditis elegans, a single gene in Drosophila and multiple genes in vertebrates. In C. elegans, the gene F10E7.4 encodes for five TSR""s in addition to the FS1 and FS2 domains (Higashijima et.al., Dev. Biol. 192:211-227, 1997). In Drosophila, the family member termed M-spondin (mspo) contains the FS1 and FS2 domains and a single TSR (Umemiya et al., Dev. Biol. 186:165-176, 1997). The M-spondin gene encodes a secreted protein that is localized at the muscle attachment sites and seems to function as an extracellular matrix protein that supports musclexe2x80x94apodeme attachment. The family members in vertebrates include genes isolated from zebrafish (Mindin1 and Mindin2, F-spondin1, and F-spondin2), rat F-spondin, Xenopus F-spondin and rat Mindin. Mindin1 and Mindin2 are closely related to each other and have a gene structure similar to that of Drosophila M-spondin. Both Mindin1 and Mindin2 genes encode for a single TSR in addition to the FS1 and FS2 domains (Higashijima et.al., Dev. Biol. 192:211-227, 1997). Zebrafish F-spondinl and F-spondin2, rat F-spondin (Klar et al., Cell 69:95-110, 1992) and Xenopus F-spondin (Altaba et al., Proc. Natl. Acad. Sci. USA 90:8268-8272) genes all have similar structures, encoding six copies of the TSR""s in addition to the FS1 and FS2 domains. In vertebrates, the Mindin/F-spondin superfamily can be classified into two groups: those genes closely related to the original rat F-spondin and Mindin genes and those genes closely related to the Drosophila M-spondin gene. Both vertebrate Mindin and F-spondin genes code for proteins that are primarily expressed by the floor plate of the neural tube during embryonic development.
Recently, a single F-spondin related gene, AmphiF-spondin, has been isolated from amphioxus (Shimeld, S. M., Mol. Biol. Evol. 15(9): 1218-1223,1998). Based on molecular phylogenetics, AmphiF-spondin is closely related to a particular subgroup of vertebrate F-spondin genes that encode six TSR""s. AmphiF-spondin encodes three TSR""s and two fibronectin type III repeats, one of which has strong identity to a fibronectin type III repeat from Deleted in Colorectal Cancer (DCC). The expression of the protein is found through most of the central nervous system and is not confined to the midline as described for the vertebrate Mindin and F-spondin proteins.
These data suggest that extracellular matrix proteins, such as the novel RG1 protein, which is a homologue of the Mindin/F-spondin superfamily, would be good candidates for use in diagnosis of cancer and therapeutic intervention.
The present invention provides a polynucleotide sequence which uniquely encodes a novel protein designated herein as RG1. The RG1 polypeptide shows homology to the rat Mindin extracellular matrix protein. It contains a hydrophobic signal sequence at the N-terminus, two spondin domains (FS1 and FS2), and a thrombospondin type 1 repeat at its C-terminus. RG1 shows 89.7% similarity to rat Mindin. The polynucleotide sequence, designated herein as rg1, and described herein in FIG. 1 (SEQ ID NO. 1), encodes the amino acid sequence for RG1, which is shown in FIG. 2 (SEQ ID NO. 2).
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel proteins with homology to the Mindin family of extracellular matrix proteins, as shown by comparison of the amino acid sequence set out in FIG. 2 (SEQ ID NO: 2) and known amino acid sequences of other extracellular matrix proteins.
It is a further object of the invention, moreover, to provide polynucleotides that encode such polypeptides, particularly polynucleotides that encode the polypeptide designated herein as RG1.
In accordance with this aspect of the invention there are provided isolated polynucleotides encoding RG1, including mRNAs, cDNAs, and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of polynucleotides that encode variants of the polypeptide designated herein as RG1.
In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as RG1 as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
Among the particularly preferred embodiments of this aspect of the invention are variants of RG1 encoded by naturally occurring allelic variants of the rg1 polynucleotide.
It is another object of the invention to provide a method of producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment of this aspect of the invention there are provided methods of producing the aforementioned RG1 polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived RG1-encoding polynucleotide under conditions for expression of human RG1 in the host and then recovering the expressed polypeptide.
In accordance with another object of the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for inter alia research, biological, clinical and therapeutic purposes.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia, for assessing RG1 expression in cells by determining RG1 polypeptides or RG1-encoding mRNA; and assaying genetic variation and aberrations, such as defects, in rg1 genes.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided probes that hybridize to rg1 sequences.
It is a further object of the invention to provide antibodies which are highly selective for RG1 polypeptides, or fragments thereof, and which may be employed in a method for diagnosis and/or detection of RG1 expression, which may be associated with prostate cancer. In accordance with certain preferred embodiments of this aspect of the invention, antibodies are labeled in such a way as to produce a detectable signal. Particularly preferred would be an antibody labeled with a radiolabel, an enzyme, a chromophore or a fluorescer.
In a further aspect of the invention there are provided antibodies which are conjugated to a therapeutic agent for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. Particularly preferred in this regard are therapeutic agents which are cytotoxic. In certain preferred embodiments in this regard is administration of such conjugated antibodies to a human patient for treatment of a disease state characterized by RG1 activity or expression such as prostate cancer.
In a further aspect of the invention, peptides and anti-idiotypic antibodies are provided which can be used to stimulate an immune response.
In a further aspect of the invention there are provided ribozymes and polynucleotides complementary to rg1 polynucleotides (i.e. antisense polynucleotides) for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. Particularly preferred in this regard is administration of antisense molecules to a human patient for treatment of a disease state, such as prostate cancer or benign prostatic hyperplasia, which is alleviated by decreasing the level of RG1 activity.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.